The PI seeks continued funding to improve our understanding of the molecular pharmacology of inhaled anesthetic (IA) action through a detailed characterization of anesthetic binding. We have determined the molecular architecture of three IA binding sites in mammalian proteins, and the distribution of binding at both the brain regional and protein level. IA binding to protein relies on internal cavity volume, hydrophobic and weak van der Waals forces, whereas full electrostatics reduce affinity. Maximum association constants of approximately 1000/M indicate low stringency of sites, and therefore suggests many widely distributed targets. This was confirmed using photolabeling in brain sections and 1D SDS-PAGE. These data suggest the need for a pharmacodynamic model of action that incorporates multiple targets, but further evidence is required. In the proposed aim 1, we will characterize binding site architecture using systematic mutagensis, spectroscopy, calorimetry and crystallography in a new surrogate peptide model. This aim will test the hypothesis that the maximum IA binding affinity is approximately 1mM, that hydrophobicity (internal cavity volume) dominates the binding energetics, but that sufficient stringency exists to select for different classes of IA. These data will then serve as a basis for PDB data mining. The 2nd aim will use IA photolabeling, 2D gels and MALDI-MS to refine the search for binding targets from rat and human CNS tissue. The hypothesis underlying this aim is that a minority of brain proteins bind IA specifically, and that different IAs will exhibit unique selectivity. These experiments will identify binding targets, and provide affinity and stoichiometry values. Although affinity will be similar in these targets, we hypothesize that binding stoichiometry will vary more.. Stoichiometry may provide the selectivity that is missing when considering only affinity, i.e., functional targets may be a subset of binding targets. In an initial attempt to weight the binding data for pharmacological relevance, we will quantitatively examine the expression pattern of that group of proteins from aim 2 that bind IA specifically in animal and cell models exposed to the same IA. At the end of this cycle we expect to know the structural basis for IA affinity, the distribution of IA binding sites within and between resolvable mammalian CNS proteins, and the identification of novel IA targets for further study.